RESUMO
The malarial parasite Plasmodium vivax causes disease in humans, including chronic infections and recurrent relapses, but the course of infection is rarely fatal, unlike that caused by Plasmodium falciparum. To investigate differences in pathogenicity between P. vivax and P. falciparum, we have compared the subtelomeric domains in the DNA of these parasites. In P. falciparum, subtelomeric domains are conserved and contain ordered arrays of members of multigene families, such as var, rif and stevor, encoding virulence determinants of cytoadhesion and antigenic variation. Here we identify, through the analysis of a continuous 155,711-base-pair sequence of a P. vivax chromosome end, a multigene family called vir, which is specific to P. vivax. The vir genes are present at about 600-1,000 copies per haploid genome and encode proteins that are immunovariant in natural infections, indicating that they may have a functional role in establishing chronic infection through antigenic variation.
Assuntos
Genes de Protozoários , Plasmodium vivax/genética , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Cromossomos Artificiais de Levedura , DNA de Protozoário , Biblioteca Gênica , Variação Genética , Humanos , Malária Vivax/parasitologia , Família Multigênica , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Plasmodium vivax/imunologia , Plasmodium vivax/patogenicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Pseudogenes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TelômeroRESUMO
Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.
Assuntos
Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania mexicana/efeitos dos fármacos , Animais , Resistência a Medicamentos , Amplificação de Genes , Leishmania mexicana/citologia , Leishmania mexicana/enzimologia , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The catalytic properties of ornithine decarboxylase (ODC) from Leishmania mexicana as well as the interaction with its cofactor pyridoxal 5'-phosphate (PLP) and the irreversible inhibitor alpha-difluoromethylornithine (DFMO) have been studied using partially purified preparations of the enzyme obtained from parasite promastigotes. Leishmania extracts prepared in the presence of saturating concentrations of PLP yielded an enzyme considerably more resistant to heat inactivation and with a three-fold higher activity than the ODC obtained without the addition of cofactor. The complete removal of PLP by treatment with hydroxylamine yielded the apoenzyme which shows an absolute requirement for PLP to recover its enzymatic activity. The Km values for L-ornithine and PLP were 0.7 mM and 25 microM, respectively, while Ki for DFMO was 0.2 mM. The restoration of ODC activity from apoenzyme and cofactor seems to involve time and temperature-dependent activation processes. L. mexicana ODC has an apparent molecular mass of 240 +/- 20 kDa.
Assuntos
Eflornitina/farmacologia , Leishmania mexicana/enzimologia , Ornitina Descarboxilase/metabolismo , Fosfato de Piridoxal/farmacologia , Animais , Apoenzimas/metabolismo , Catálise , Cromatografia em Gel , Ativação Enzimática/efeitos dos fármacos , Reativadores Enzimáticos/farmacologia , Temperatura Alta , Hidroxilamina , Hidroxilaminas/farmacologia , Peso Molecular , Ornitina Descarboxilase/química , Inibidores da Ornitina DescarboxilaseRESUMO
Repeated treatments of Leishmania mexicana promastigote cultures with a-difluoromethylornithine could not block proliferation when the parasite was grown in a rich medium. Although the irreversible inhibitor of ornithine decarboxylase was able to abolish the enzymatic activity under these conditions, polyamine depletion was only partial probably due to the uptake of these substances from the external medium. Conversely, when Leishmania was cultivated in a defined medium essentially free of polyamines, a-difluoromethylornithine was able to decrease the growth rate and proliferation was arrested after several passages in the presence of the drug. Parasite multiplication could be resumed by addition of exogenous polyamines, and a strict correlation between Leishmania promastigote growth and intracellular levels of spermidine was observed.
Assuntos
Eflornitina/farmacologia , Leishmania mexicana/fisiologia , Poliaminas/metabolismo , Animais , Cinética , Leishmania mexicana/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Poliaminas/farmacologia , Reprodução/efeitos dos fármacosRESUMO
Studies on the decarboxylation of ornithine in Leishmania mexicana have shown that this activity corresponds to a true ornithine decarboxylase rather than to an oxidative decarboxylation or aminotransferase reaction, both of which also give rise to the release of CO2. The stoichiometric relationship between substrate and products has indicated that extracts of L. mexicana were able to catalyse the formation of an unknown compound besides putrescine and CO2. The addition of cycloheximide to cultures of L. mexicana allowed us to demonstrate that ornithine decarboxylase degradation in vivo was extremely slow in this parasite. This remarkable stability of the enzyme is only comparable to that found in Trypanosoma brucei and contrasts with the high turnover rate of ornithine decarboxylases of different mammalian cells.